The bacterial groups present in caecal content were characterized by means of the fluorescent in situ hybridization (FISH) technique coupled to flow cytometry (FCM) analysis using group- or genus-specific fluorochrome-conjugated probes (Table 1 ), which target the bacterial 16S rRNA (Sigma-Aldrich, Madrid, Spain), as previously established [29 (link),30 (link),31 (link)]. FCM analysis (FacsAria SORP sorter, BD, San José, CA, USA) was carried out in the flow cytometry unit of the Scientific and Technological Centres of the University of Barcelona (CCiT-UB). Analysis was performed using FlowJo v10 software (Tree Star, Inc., Ashland, OR, USA). Microbiota data were expressed as total bacteria counts/g caecal content, as well as the relative percentage of each bacterial group (the sum of all percentages of each studied probe was considered as 100%).
Facsaria sorp sorter
Manufactured by BD
Sourced in United States
The FacsAria SORP sorter is a cell sorting instrument designed for high-performance flow cytometry applications. It features advanced optics and sorting capabilities to enable precise cell identification and separation.
Automatically generated - may contain errors
Lab products found in correlation
Flow check fluorospheres, by Beckman Coulter (3 mentions)
Erec482, by Merck Group (2 mentions)
Lab158, by Merck Group (2 mentions)
Fluorescein isothiocyanate (fitc), by Abcam (2 mentions)
Facsaria 2, by BD (2 mentions)
Purelink rna micro kit, by Thermo Fisher Scientific (2 mentions)
Dulbecco phosphate buffered saline (dpbs), by Thermo Fisher Scientific (2 mentions)
Live dead aqua dead cell stain kit, by Thermo Fisher Scientific (2 mentions)
Facs stain buffer, by BD (2 mentions)
Anti cd16 cd32 fc block clone 2.4g2, by BD (2 mentions)
Diva 6, by BD (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
Bif164, by Merck Group (1 mentions)
Cytometric bead assay rat soluble protein flex set, by BD (1 mentions)
Propidium iodide staining, by Merck Group (1 mentions)
Lsrfortessa, by BD (1 mentions)
Attune nxt flow cytometer, by Thermo Fisher Scientific (1 mentions)
Annexin 5 staining kit, by Thermo Fisher Scientific (1 mentions)
Cyan immunocytometry system, by Agilent Technologies (1 mentions)
12 protocols using facsaria sorp sorter
1
Profiling Caecal Microbiota by FISH-FCM
2
T-cell Subpopulation Sorting by CD146
3
Targeted Quantification of Gut Bacteria by FISH-FCM
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The previous effects observed by the polyphenols interventions on particular bacteria groups, such as Clostridium coccoides/Eubacterium rectale, Bifidobacterium and Lactobacillus/Enterococcus, led us to evaluate the changes induced by exercise only on these populations. For that, sequencing was initially discarded and targeted fluorescence in situ hybridization coupled to flow cytometry (FISH-FCM) as previously used in our group [48 (link)], was used. Group-specific fluorochrome-conjugated probes (Erec482 5′-GCTTCTTAGTCARGTACCG, Bif164 5′-CATCCGGCATTACCACCC, and Lab158 5′-GGTATTAGCAYCTGTTTCCA) (Sigma-Aldrich, Madrid, Spain), which hybridize the bacterial 16S RNA of each particular group were used. Data were acquired by a FacsAria SORP sorter (BD Biosciences, San Diego, CA, USA) in the Flow Cytometry Unit (FCU) of the Scientific and Technological Centers of the University of Barcelona (CCiTUB) and the analysis was performed with FlowJo v.10 software (Tree Star Inc., Ashland, OR, USA). The results were normalized by total bacteria, which was detected adding propidium iodide (PI; Sigma-Aldrich) prior to the FCM acquisition. Commercial Flow CheckTM Fluorospheres (Beckman Coulter, Miami, FL, USA) were used to assess the total counts of bacteria.
4
Cytokine Profiling in Spleen Cells
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The cytokine concentration in supernatants from stimulated spleen cells and from GW samples was measured. Antiviral and Th1 (IFNγ), Th2 (IL-4), anti-inflammatory and regulatory (IL-10), and pro-inflammatory (TNFα) cytokines were evaluated. Molecule determinations were performed using a BD™ Cytometric Bead Assay Rat Soluble Protein Flex Set (BD Biosciences, Madrid, Spain) as detailed in previous studies (53 (link)). A FacsAria SORP sorter (BD, San José, CA, USA) from the cytometry service of the Scientific and Technological Centers of the University of Barcelona (CCiT-UB) was used. Data analysis was performed using the FlowJo 10.0.7 software (Tree Star, Inc., Ashland, OR, USA).
5
Quantifying IgA-Coated Bacteria by FCM
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The proportion of IgA-coated bacteria (IgA-CB) in CC was determined by FCM as previously described [48 (link)]. The CC homogenate was stained with rabbit anti-rat Ig polyclonal antibody conjugated to fluorescein isothiocyanate (FITC) (Abcam, Cambridge, UK). Bacteria were gated in a FacsAria SORP sorter (BD Biosciences) after PI staining (Sigma-Aldrich) and according to their forward (FSC) and side scatter (SSC) characteristics. Analysis was performed in the FCU of the CCiTUB using the FlowJo v.10 software.
6
Isolation of CD31+ endothelial cells
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We obtained CD31+ endothelial cells by cell sorting, essentially as previously reported [14 (link)]. Unspecific binding was blocked by incubation for 10 min with anti CD16/CD32 (Fc block, clone 2.4G2; #553142, BD Pharmingen, RRID:AB_394657) in FACS buffer at 4 °C. Live/dead Aqua Dead Cell stain kit (#L34957, Thermo Fisher Scientific) was used to determine the viability of cells. Cells were incubated with the following primary antibodies during 30 min at 4 °C: CD11b (clone M1/70, APC-Cy7, #557657, BD Pharmingen, RRID:AB_396772), CD45 (clone 30-F11, FITC, #553080, BD Pharmingen, RRID:AB_394610), CD31 (clone 390, PE-Cyanine7, #25-0311-82, eBioscience, RRID:AB_2716949). After washing with FACS Stain Buffer (#554656, BD Biosciences), the cells were sorted in a FACSAriaII or FACSAria SORP sorter (BD Biosciences). Endothelial cells were collected in sterile DPBS (#14190-094, Thermo Fisher Scientific), centrifuged, and resuspended in lysis buffer (from PureLink™ RNA Micro Kit #12183016, Invitrogen) supplemented with 10% β-mercaptoethanol and finally snap-frozen in dry ice.
7
Quantifying Gut Microbiome Changes via FISH-FCM
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Two of the most studied bacterial groups involved in the microbiota changes induced by exercise, the Clostridium coccoides/Eubacterium rectale and Lactobacillus/Enterococcus were determined by fluorescence in situ hybridization coupled to flow cytometry (FISH-FCM) analysis using group-specific fluorochrome-conjugated probes (Erec482 5′-GCTTCTTAGTCARGTACCG and Lab158 5′-GGTATTAGCAYCTGTTTCCA, respectively) (Sigma-Aldrich), which hybridize the bacterial 16S RNA of each particular group as previously detailed (26 (link)). Data were acquired by a FacsAria SORP sorter (BD Biosciences) in the FCU of the CCiTUB and the analysis was performed with FlowJo v.10 software (Tree Star). The results were normalized by total bacteria, which was detected adding PI (Sigma-Aldrich) prior to the flow cytometry acquisition. Commercial Flow Check™ Fluorospheres (Beckman Coulter, Inc., FL, United States) were used to assess the total counts of bacteria.
8
Isolation of CD31+ endothelial cells
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We obtained CD31+ endothelial cells by cell sorting, essentially as previously reported [14 (link)]. Unspecific binding was blocked by incubation for 10 min with anti CD16/CD32 (Fc block, clone 2.4G2; #553142, BD Pharmingen, RRID:AB_394657) in FACS buffer at 4 °C. Live/dead Aqua Dead Cell stain kit (#L34957, Thermo Fisher Scientific) was used to determine the viability of cells. Cells were incubated with the following primary antibodies during 30 min at 4 °C: CD11b (clone M1/70, APC-Cy7, #557657, BD Pharmingen, RRID:AB_396772), CD45 (clone 30-F11, FITC, #553080, BD Pharmingen, RRID:AB_394610), CD31 (clone 390, PE-Cyanine7, #25-0311-82, eBioscience, RRID:AB_2716949). After washing with FACS Stain Buffer (#554656, BD Biosciences), the cells were sorted in a FACSAriaII or FACSAria SORP sorter (BD Biosciences). Endothelial cells were collected in sterile DPBS (#14190-094, Thermo Fisher Scientific), centrifuged, and resuspended in lysis buffer (from PureLink™ RNA Micro Kit #12183016, Invitrogen) supplemented with 10% β-mercaptoethanol and finally snap-frozen in dry ice.
9
Evaluating Gut Immune Response via IgA-Coated Bacteria
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The proportion of IgA-coated bacteria (IgA-CB) was evaluated as an indirect immune biomarker of the intestinal immune response against pathogens (35 (link)). IgA-CB in CC was determined by flow cytometry as previously described (26 (link)), with minor modifications. In the current study, the CC homogenate was stained with rabbit anti-rat Ig polyclonal antibody conjugated to fluorescein isothiocyanate (FITC) (Abcam, Cambridge, United Kingdom). Bacteria were gated in a FacsAria SORP sorter (BD Biosciences, Madrid, Spain), after propidium iodide (PI) staining (Sigma-Aldrich, Madrid, Spain) and according to their forward (FSC) and side scatter (SSC) characteristics. Analysis was performed in the Flow Cytometry Unit (FCU) of the Scientific and Technological Centers of the University of Barcelona (CCiTUB) using the FlowJo v.10 software (Tree Star, Inc., Ashland, Oregon, United States).
10
Quantifying FISH and IgA-coated Bacteria
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For FISH and IgA-coated bacteria quantification, FCM analysis was performed using a FacsAria SORP sorter (BD, San José, CA, USA) as previously described [5] . Commercial Flow Check TM Fluorospheres (Beckman Coulter, Inc. FL, USA) were used to determine total counts combined with PI. Analysis was performed using Flowjo v7.6.5 software (Tree Star, Inc.). Microbiota composition results are expressed as the log 10 of specific probe labeled bacteria counts/g of feces in each sample. Moreover, the Firmicutes to Bacteroidetes (F/B) ratio was calculated taking into account the analyzed bacterial groups belonging to the Firmicutes phylum (those hybridized by Chis150, Erec482, Lab158, Staphy and Strept probes) and those belonging to the Bacteroidetes phylum (those hybridized by the Bac303 probe). IgA-coated bacteria results are expressed as the percentage of bacteria coated with IgA with respect to the total bacteria.
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